1. Field of Invention
The present invention relates generally to methods and apparatus for acquiring and quantifying data, and more particularly to a method and apparatus for acquiring and quantifying high-resolution, three-dimensional (3D) data. This invention is particularly well-suited for classification or typing based on quantitative data acquired from biological cells and macromolecules using atomic force microscopy.
2. Discussion of the Related Art
Determining the morphology of biological cells and macromolecules (hereinafter biological structures) is important for a number of tasks, including (1) cell typing (e.g., typing blood cell into red blood cells, lymphocytes, platelets, etc.) and (2) classifying cells and other biological structures into normal and abnormal cells (e.g., classifying benign, premalignant, and malignant cells). Morphological determination of biological structures has traditionally been accomplished using microscopy, first with light microscopy and later with electron microscopy. More recently, local sensitive force detectors, such as atomic force microscopes (AFMs), have been used for obtaining data measurements.
Confocal microscopes have the highest resolution of all light microscopes, but have a lateral (X-Y) resolution of only about 200 nm and a vertical (Z) resolution of only about 650 nm. In contrast, electron microscopes (EMs) and AFMs have a much higher resolution. Specifically, AFMs have a lateral (X-Y) resolution of 1 nm. Theoretically, AFMs have a vertical (Z) resolution of 0.01 nm (i.e., 0.1 angstrom (xc3x85)). Because of environmental noise, however, in practice AFMs have a Z-axis resolution of around 1.0 xc3x85. In comparison, AFMs provide two orders of magnitude better resolution than light microscopes and comparable resolution to EMs.
While the lateral resolution of EMs is sufficient to discriminate subtle surface features, EMs have many practical limitations for use as an analytical tool for biological material. The main problem is that the typical biological material has low electron density and conductivity. Therefore, to permit good visualization and to prevent sample damage from electronic radiation, EM samples are typically dried and metal-coated. Additionally, EM samples often require sectioning and chemical fixation. Consequently, sample preparation for EM is time-consuming. Furthermore, due to sample preparation requirements, EMs cannot be used to study biologically active samples.
In contrast, imaging with an AFM is relatively fast because the sample does not require drying, sectioning, metal coating or chemical fixing. Thus, AFMs may be used with samples that require very little sample preparation, including (1) samples that are biologically active and (2) samples in both ambient air (including dried samples) and liquid.
The typical AFM has a probe comprising (1) a flexible cantilever and (2) a tip disposed on the free end of the cantilever. Interactions between the tip and the sample influence the motion of the cantilever, and one or more parameters of this influence are measured to generate data representative of one or more properties of the sample. AFMs can be operated in different modes including contact mode, TappingMode, light TappingMode, (Tapping and TappingMode are trademarks of Digital Instruments, a Division of Veeco Instruments Inc.), and non-contact mode. In contact mode, the cantilever is not oscillated, and cantilever deflection is monitored as the tip is dragged over the sample surface. In TappingMode, the cantilever is oscillated mechanically at or near its resonant frequency so that the probe tip repeatedly taps the sample surface, thus reducing the tip""s oscillation amplitude. The change in oscillation amplitude indicates proximity to the sample surface and may be used as a signal for feedback. Changes in other oscillation parameters, such as phase, may also be monitored. U.S. patents relating to Tapping and TappingMode include numbers 5,266,801; 5,412,980; and 5,519,212, by Elings et al., all of which are hereby incorporated by reference. In the non-contact mode, attractive interactions between the tip and the sample (commonly thought to be due to Van der Waals"" attractive forces) shift the cantilever resonance frequency when the tip is brought within a few nanometers of the sample surface. These shifts can be detected as changes in cantilever oscillation resonant frequency, phase, or amplitude, and can be used as a feedback signal for AFM control.
Whether operating in contact mode, TappingMode, or non-contact mode, feedback is typically used during AFM scanning to adjust the vertical position of the probe relative to the sample so as to keep a probe operational parameter, such as the tip-sample interaction, constant. A measurement of surface topography or another sample characteristic may then be obtained by monitoring a signal such as the voltage used to control the vertical position of the scanner. Alternatively, independent sensors may monitor the position of the tip during scanning to obtain a map of surface topography or another measured sample characteristic. Measurements can also be made without feedback by monitoring variations in the cantilever deflection as the probe moves over the surface. In this case, recording the cantilever motion while scanning results in an image of the surface topography in which the height data is quantitative. Additionally, the positioning of the AFM probe can be enhanced by compensating for drift. U.S. patents relating to drift compensation include 5,081,390 and 5,077,473 by Elings et al., both of which are hereby incorporated by reference.
As described above, the AFM typically provides up to 1 xc3x85 resolution for the Z-axis and 1 nanometer resolution for the X- and Y-axes for samples in air or liquid. Traditionally, AFM data recorded from all three axes of a biological structure are displayed as computer generated, space filling images with height-encoded scaling. These images are analyzed directly from the computer-rendered two-dimensional (2D) or three-dimensional (3D) images. Additionally, the most widely used practice for further analysis of AFM results is simply a subjective description of the AFM image, such as height, depth, width, distance between features, Fourier averaging, volume, surface area roughness, force versus distance curves, and 2D section profiles.
While many measurements exist for AFM imaging, AFMs are currently not used as a clinical diagnostic tool for several reasons. One reason is that samples cannot be marked for differentiation by AFMs. For instance, AFMs cannot differentiate a dyed sample from an undyed sample, whereas confocal microscopes can. One example of dyeing samples for differentiation purposes is employing dye exclusion to count viable cells. Viable cells are impermeable to naphthalene black, trypan blue, and a number of other dyes. After these dyes are added to cells, the cells can be examined by light microscopes to determine the proportion of viable cells to non-viable cells. In contrast, AFMs are not typically used to detect such dyes.
Another reason that atomic force microscopy is currently not used as a clinical diagnostic tool is that there is a lack of objective methods to analyze the AFM data beyond displaying a space filling image of the biological structure based on two or three axes of the biological structure. In rare instances, proposals have been made to plot the AFM data as a function of another experimental variable, resulting in more unique graphical forms. For instance, Radmacher et al., xe2x80x9cDirect Observation of Enzyme Activity with the Atomic Force Microscope,xe2x80x9d Science, Vol. 265, Sep. 9, 1994 (Radmacher), proposes placing an AFM tip in stationary mode on an enzyme, and plotting the height fluctuations that the enzyme undergoes during an enzymatic reaction versus time in seconds. Another proposed use is disclosed in Allen et al., xe2x80x9cExtent of Sperm Chromatin Hydration Determined by Atomic Force Microscopy,xe2x80x9d Molecular Reproduction and Development, Vol. 45, 1996 (Allen). Allen plots AFM-determined volumes of sperm nuclei as a function of hydration level in the AFM fluid imaging chamber. While Radmacher and Allen both disclose generating graphical forms by plotting AFM data as a function of another experimental variable, they, and others, neither state nor imply (1) how to generate and use a single, height-dependent (e.g., Z-axis dependent) plot, which is derived entirely from a single AFM image of a biological structure, or (2) how to compare the height-dependent data, or any other data obtained from unknown biological structures to standard data generated from known biological structures that are stored in a computer database.
With this lack of methods to analyze AFM data, what is needed is a method to objectively analyze data of interest and to compare AFM data from an unknown biological structure to data from known biological structures. These comparisons are desirable for several tasks including (but not limited to) cancer diagnosis, which is based on comparing an unknown cell to benign, premalignant, and malignant cells. This comparison is based on morphological (or shape) differences between cancer, premalignant, and benign cells. Thus, distinct morphological features are diagnostic for malignant and premalignant states. One reason for this morphological change is because the nuclei expand as the cell undergoes rapid division. Nuclei of malignant cells can also have irregular shapes. The overall cell morphology (as opposed to just the nuclei morphology) can also change with malignancy. Additionally, the morphology of premalignant cells also varies between benign and cancer cells. Traditionally, distinct morphological features which are diagnostic of malignant and premalignant cells are typically detected by manual observations using light microscopy.
Some automation of light microscopy has occurred. For example, U.S. Pat. No. 5,287,272, (the ""272 patent) to Rutenberg et al. discloses an apparatus and method for automated cytological specimen classification using an automated light microscope and associated image processing circuitry. The ""272 patent uses a multi-step classification process that includes an optional last step of having a human operator examine the cells remaining after the initial classifications. Another example is U.S. Pat. No. 5,740,269, to Oh et al., which discloses an automated light microscope that obtains features from an image of a biological specimen slide, computes feature variations, and classifies biological specimens. However, the automation of light microscopy is typically limited by several factors including (1) the low level of resolution that light microscopy provides and (2) the reliance in many methods on having a human operator subjectively compare benign cells to malignant or premalignant cells, as opposed to a totally objective comparison. Accordingly, it would be desirable to have a high-resolution microscopy method and apparatus that objectively compares data from unknown biological structures to data from known biological structures so that an unknown biological structure can be compared and classified or typed.
It would also be desirable to have such a method and apparatus that additionally could be used in conjunction with light microscopy. Light microscopes and high-resolution microscopes (e.g., AFMs ) have been previously combined. For example, U.S. Pat. No. 5,689,063 to Fujiu et al. discloses the use of an optical microscope to confirm the scanning position of an AFM by using the images from both microscopes. Another example is U.S. Pat. No. 5,360,977 to Onuki et al., which discloses the use of an optical microscope to observe a sample being measured by an AFM. While both of these patents combine optical microscopes with AFMs, neither patent discloses how to calculate the high-resolution data acquired corresponding to a single axis within a biological structure.
An object of the invention is to provide a method and apparatus for acquiring and quantifying, in an objective format, data from two axes of a biological structure as a function of scan position along a previously defined third axis.
In accordance with a first aspect of the invention, this object is achieved by providing a biological structure, scanning the biological structure with a local sensitive force detector, and acquiring and quantifying data from points along a single axis of a biological structure.
Another object of the invention is to provide a method for generating standard data from libraries of acquired and quantified data for subsequent comparison to data acquired and quantified from unknown biological structures.
In accordance with this aspect of the invention, the method acquires and quantifies data from along a single axis of a biological structure and corresponding data from known biological structures. These standard data are created by averaging or otherwise analyzing data from multiple biological structures from a single class of known biological structures (e.g., malignant cervical cells). Then, the averaged data can be plotted on a two-dimensional plot. The data can also be displayed and also be archived.
Another object of the invention is to provide an objective method for classifying and typing biological structures.
In accordance with this aspect of the invention, the method can be used to acquire and quantify data from along a single axis of an unknown biological structure. The data from the unknown can then be compared to the standard data from known biological structures. The results of this comparison can be used to classify the unknown as belonging to one of the known classes or types. For example, the method can be used to classify cells as being benign, premalignant, or malignant cells. Additionally, the method can be used to classify human sperm nuclei into one of the nine or more abnormal human sperm nuclei subtypes. The method can also be used to type biological structures, such as blood cells, by typing and counting the different kinds of blood cells. From these countings of different kinds of blood cells, ratios of specific types of blood cells can be determined.
Another object of the invention is to provide multiple methods of quantifying the data from along a single axis of a biological structure.
In accordance with this aspect of the invention, the method provides at least two embodiments for data handling. First, the raw data (i.e., the number of data points within a particular computer file corresponding to a region on the sample surface) is acquired and quantified. Second, the surface area corresponding to one angstrom (xc3x85) thick theoretical xe2x80x9cslicesxe2x80x9d is calculated for horizontal planar increments through a single axis.
Another object of the invention is to provide an objective apparatus and method to acquire data having more than two orders of magnitude higher resolution than data acquired from digitized light microscopy.
In accordance with this aspect of the invention, the method provides 1.0 xc3x85 resolution for the Z-axis and 1.0 nanometer resolution for the X- and Y-axes. Better resolution could be provided with a reduction in ambient noise.
Still another object of the invention is to provide an objective method that has little or no sample preparation requirement.
In accordance with still another aspect of the invention, the method is capable of operating on biological structures that do not require sample preparation, including: chemical fixing, metal coating, drying, hardening, and time consuming sectioning.
Still another object of the invention is to provide an objective method that permits analysis of structures in ambient air or in liquid (including intact and/or active biological structures) and analysis of dried samples (including i.e., Pap smears).
In accordance with still another aspect of the invention, the method uses samples that are either in ambient air or in liquid. Additionally, the method can also use dried samples.
Still another object of the invention is to provide an objective method for use on samples conventionally prepared for use with light microscopy and to provide a method for use in tandem with light microscopy.
In accordance with this aspect of the invention, the method preferably uses samples deposited on a substrate such as a glass slide. These materials permit transmitted light microscopy to be performed in tandem with AFM on the same sample. Additionally, samples may be deposited on any transparent substrate, such as mica. For example, the method could be used for Pap smears that have been prepared for light microscopy.
Still another object of the invention is to provide a local sensitive force detector capable of acquiring quantified data from along a single axis of a biological structure into a format suitable for plotting data on a two-dimensional graph, such as a histogram.
In accordance with still another aspect of the invention, this object is achieved by providing a local sensitive force detector that includes a probe, a detection device, and a computer. The probe is configured to react to a surface of a biological structure. The detection device monitors operation of the probe and is capable of detecting a position of the probe, which changes with respect to the surface of the biological structure. The computer is configured to (1) acquire data from along a single axis of a biological structure and (2) quantify the data from the single axis of the biological structure as a function of position along the axis.
The local sensitive force detector preferably comprises an atomic force microscope (AFM).